Cartridge And Device For Analyzing Biological Samples Using Temperature-Controlled Biological Reactions

ABSTRACT

The cartridge according to the invention for analysing biological samples comprises:
         a reaction chamber and a biochip mounted in the reaction chamber,   a filling nozzle connected so as to communicate with the reaction chamber, and   a compensation chamber connected so as to communicate with the reaction chamber,   wherein the reaction chamber, the compensation chamber and all lines connected thereto form a chamber sealed as far as the filling nozzle, wherein
 
the filling nozzle forms a free passage to the reaction chamber from outside the cartridge, and a stopper is provided which fits positively and tightly in the filling nozzle in such a way that, when pressed in over a certain distance, fluid is displaced from the filling nozzle towards the reaction chamber.

The invention relates to a cartridge and an apparatus for analysingbiological samples using temperature-controlled biological reactions.

A biochip has a generally flat substrate with different catchermolecules located at predetermined points, the spots, on the surface ofthe substrate. A sample substance provided with a marking reacts withcertain catcher molecules on the lock and key principle. The catchermolecules are generally comprised of DNA sequences (see e.g. B. EP 373203 B1) or proteins. Such biochips are also known as arrays or DNAarrays. The markings are often fluorescent markers. The fluorescenceintensity of the individual spots is determined by an optical reader.This intensity correlates with the number of marked sample moleculesimmobilised by the catcher molecules.

WO 2005/108604 A2 discloses a heatable reaction chamber for processing abiochip. This reaction chamber has an elastic membrane. Mounted on themembrane is a silicon biochip. Provided as heating device is anickel-chromium thin-film conductor path. Such nickel-chromium thin-filmconductor paths have high electrical resistance and a correspondinglyhigh heat output. In addition to the conductor path for the resistanceheating, a further conductor path is provided for temperaturemeasurement.

In this known reaction chamber, one housing wall is designed asmembrane, so that the biochip may be pressed against a cover glass lyingopposite the membrane by means of a stopper. By this means, a reactionfluid in the reaction chamber is displaced from the surface of thebiochips and does not disturb the optical detection. A seal is providedbetween the membrane and the cover glass. The sample fluid is poured inby means of a filling cannula, which is pushed through the seal. Inconnection with stoppers, surplus sample fluid is drawn out of thereaction chamber by means of a pressure equalisation cannula.

In U.S. Pat. No. 5,759,846 and U.S. Pat. No. 6,130,056 a reactionchamber for holding biological tissues is described in each case. Thereaction chamber contains a flexible circuit board with electrodes. Bypressing together the tissue and the flexible circuit board, anelectrical contact can be made between the biological tissue and theelectrodes of the flexible circuit board, so that an electrical pickupmay be made directly at the biological tissue.

Described in DE 10 2005 09 295 A1 is a chemical reaction cartridge withseveral chambers. By rolling a roller over the surface of the cartridge,fluids may be moved from one chamber to another. Also provided is ametal bar which may be used to exert pressure, vibration, heat, coldnessor the like on the cartridge, in order to accelerate the chemicalreaction in the cartridge.

From K. Shen et al. Sensors and Actuators B 105 (2005), pages 251-258,“A microchip-based PCR device using flexible printed circuittechnology”, the use is known of a flexible circuit board for theheating of a reaction chamber, which is provided for a PCR process. Thereaction chamber comprises a glass plate, a frame and a plastic cover.On the inside of the glass plate the flexible circuit board is fixedeither directly by means of an adhesive bond or by means of a copperchip lying in between. On account of the good thermal properties of theflexible circuit board, heating rates of 8° C./s have been obtained.Formed on the flexible circuit board is a conductor path which is usedboth for heating and also for temperature measurement. The heating takesplace during a “heating state” and the measurement during a “sensingstate”, with these states being offset in time.

Described in WO 2007/051863 A2 is a reaction chamber, in which a biochipmay be processed. The reaction chamber has two opposite walls, betweenwhich the biochip is located. One of the two walls is transparent, whichis effective both for the exciting radiation and for the signals emittedby the biochip. At least one of the two walls is so movable that thespace between the biochip and the transparent wall may be compressed, bywhich means the sample solution between them may be displaced.

US 2004/0047769 A1 and JP 2002-365299 A respectively disclose a pocketof plastic material used to hold blood. The blood may be treated foranalysis using a DNA array. The DNA array is integrated in the pocket.By means of rollers, the blood and a sample solution in the pocket aredisplaced towards the DNA array and a waste products area behind it. TheDNA array may be read in a conventional manner.

After the blood has once been put in this bag, the aim is for allreactions to take place in the bag, without the blood and the solutionsit contains leaving the bag and coming into contact with theenvironment. In this way it is possible to avoid contamination by bloodwhich may be infected.

U.S. Pat. No. 6,569,674 B1 describes an apparatus for the conduct ofbiological reactions. A reaction chamber has a microscope glass slidewhich is covered by a flexible film. Formed in the glass slide are firstand second passages which lead into the reaction chamber between thesubstrate and the flexible film. The flexible film is fastened to thesubstrate by an adhesive. Also defined in this patent is a sampletreatment chip which is in contact with the substrate and in turn has anopening, designed for opening the substrate. The film may be subjectedto pressure from a roller.

Described in International Patent Application PCT/EP2007/010298, not yetpublished, is an apparatus for the analysis of biological samples usingtemperature-controlled biological reactions. This comprises a reactionchamber to accommodate a biochip. The reaction chamber has at least onetransparent window, so that exciting light from outside may be radiatedon to the biochip and fluorescent light from the biochip may be emittedoutwards to a measuring device. A membrane forming at least one wall ofthe reaction chamber is provided and is made elastic, so that the windowand the biochip may be pressed against one another in order to displacesample solution lying between them. Provided in a filling hole is anon-return valve through which the sample material may be fed to thereaction chamber.

With this apparatus it is a drawback that, in filling, a relativelyprecisely measured sample volume must be fed into the cartridge tocreate the desired pressure. This requires a so-called “positivedisplacement pipette”, which is in a position to build up the necessaryoverpressure for the non-return valve. After the removal of the pipettethere is always, as a matter of principle, a residue of the sample aheadof the non-return valve, which may lead to contamination. The structureof this apparatus is relatively complex.

WO 02/50535 A1 discloses a cartridge for the analysis of biologicalsamples, in particular blood samples. This cartridge has fluid holdingchambers, each of which may be supplied with fluid via a common inlet,through a branching line system. The inlet is designed so that a syringemay be plugged into it. The fluid holding chambers are each connected toan air vent/fluid stopper, through which the air present in the fluidholding chambers before filling may escape.

DE 100 13 242 A1 discloses a chemical analyser with an analysiscassette. The analysis cassette is made up of a bottom substrate and atop substrate, with reaction cells formed between the two substrates.Formed on the top substrate is a guide orifice for connecting to aninjector. Also provided are air vents for connection with fine orifices,each suitably connected to end sections of fine flow passages for theremoval of air contained in them.

Described In DE 102 44 154 A1 is a carrier element for diagnostic tests,in particular to determine agglutination. This carrier element has abottom part and a top part. There is a central inlet, from which atleast two feed passages lead in each case to a reaction chamber. Fromeach reaction chamber a vent passage branches to the outside, preventingany backpressure on injection of the fluid. The air present in thereaction chambers is able to escape to the outside.

DE 295 00 587 U1 discloses a kit for blood group determination, whichhas a passage system with several passages. Provided at the end of eachpassage is a camera, described as the antibody compartment. Theindividual passages converge at a passage system distributor, whichmerges into a syringe holder. In each of the passages are valves whichform a kind of non-return valve, intended to prevent any return flow ofinjected material. The antibody compartments are sealed at least on oneside by a flexible film, and are provided on the other side with anoptical lens.

Known from DE 10 2004 023 217 A1 is a reaction module which has a rigidplate-like support with a flexible body mounted on top. Passages andchambers are formed between the two bodies. The solution is injectedusing a syringe. The needle of the syringe is injected directly into theflexible body. Such a module is designed to be sealed and may bedisposed of, so that it can be used to hold dangerous solutions. Thismodule is intended for use, amongst other things, for the detection oridentification of biopolymers such as DNA or RNA using a hybridisationunit.

The invention is based on the problem of creating a cartridge for theanalysis of biological samples using temperature-controlled biologicalreactions, which is easy to fill so that there is no risk ofcontamination from escaping sample solution and in addition the mostideal reaction conditions are provided in the reaction chamber.

The problem is solved by a cartridge with the features of claim 1.Advantageous developments are set out in the dependent claims.

The cartridge according to the invention for analysing biologicalsamples comprises:

-   -   a reaction chamber and a biochip mounted in the reaction        chamber,    -   a filling nozzle connected so as to communicate with the        reaction chamber, and    -   a compensation chamber connected so as to communicate with the        reaction chamber,        wherein the reaction chamber, the compensation chamber and all        lines connected thereto form a chamber sealed as far as the        filling nozzle, wherein        the filling nozzle forms a free passage to the reaction chamber        from outside the cartridge, and a stopper is provided which fits        positively and tightly in the filling nozzle in such a way that,        when pressed in over a certain distance, fluid is displaced from        the filling nozzle towards the reaction chamber.

By this means, the following advantages are obtained:

-   -   1. Since the filling nozzle is open, the sample solution may be        introduced easily, e.g. using a standard pipette. There is no        need for a syringe, to pierce a membrane. No pressure must be        built up in order to penetrate a valve. In filling, no force or        counterforce needs to be overcome for the sample to be inserted        in the cartridge. Filling is therefore effected without        pressure. There is no danger that part of the sample will be        spilt during filling.    -   2. The sample solution is put under pressure when the stopper is        pressed in. This increases the boiling point. As a result, even        under heating to temperatures in the range of around 100° C., no        gas bubbles occur in the sample solution, which might impair        measurements.    -   3. The air in the compensation chamber acts on the sample        solution like an elastic spring element, allowing further        displacement of the sample solution, while the restoring force        exerted on the sample solution by the air is small.        Consequently, the force which must be applied to a transparent        flexible film, bounding one side of the reaction chamber, in        order to displace the sample solution, is also small in        comparison with conventional reaction chambers with membranes.    -   4. This restoring force makes the process of displacing the        fluorescing sample solution reversible, if the force applied to        the film is varied. This makes possible realtime PCR and the        measurement of a melting curve.    -   5. The tight closing of the cartridge by the stopper rules out        any risk of contamination of the environment.

The open filling nozzle is connected via lines communicating with thereaction chamber, a filling level indicator and the compensationchamber, so that before filling, the respective normal pressure prevailsin all hollow spaces of the cartridge. On filling with the sample fluid,the filling nozzle fills up. The air in the cartridge is compressed bythe pressing in of the stopper. Since the sample chamber, thecompensation chamber and the filling nozzle are in communication withone another, the pressure in the overall container system is equalisedsimultaneously. Here the volumes are so dimensioned that the reactionchamber is completely filled with sample fluid, and an internaloverpressure of 0.3-0.5 bar and preferably around 0.4 bar is generated.

The apparatus has an open filling nozzle. This makes it possible tointroduce the sample solution using a pipette. It is not necessary touse a cannula with which, as with conventional apparatus of this kind, aseal is pierced, or to press the sample fluid through a valve underpressure.

In one embodiment of the apparatus according to the invention, the areaof the cartridge opposite the biochip is in the form of a transparentfilm. The flexible transparent film serves as a window for opticalmeasurements of the sample solution. In this variant it is advantageousthat the biochip itself need not be moved in the reaction chamber.

In one embodiment of the present invention, one side of the reactionchamber is bounded by a circuit board. The biochip is mounted directlyon the circuit board. Heating/measuring structures may be integrated inthis circuit board. Such a circuit board serves for heating andmeasurement of the sample solution.

Provided in the cartridge is a filling line section, which extends fromthe filling nozzle around the reaction chamber and leads into a boundaryzone of the reaction chamber at a distance from the filling nozzle.

The cartridge is filled with the open filling nozzle facing upwards. Thestopper is pressed into the filling nozzle, so that the sample fluid isdriven into the reaction chamber, which is filled from the bottom to thetop. By this means, air is displaced from the reaction chamber into acompensation chamber downstream of the reaction chamber. This has theadvantage that the air from the reaction chamber is completely displacedinto the compensation chamber.

For scanning, the cartridge according to the invention is arranged in anapparatus for the analysis of biological samples with the filling nozzlefacing downwards. In conducting an optical scan of the reaction chamber,a pressure of preferably 1 bar to 4 bar is created in the scanningchamber in such a way that the film fits up against the biochip and thesample fluid is displaced from the reaction chamber in the direction ofthe compensation chamber, while an air bubble remains above the samplefluid in the compensation chamber.

In the case of the apparatus for analysing biological samples, in theprocedural steps in which no optical scanning takes place and thereaction chamber is heated, a pressure is created in one scanningchamber of the apparatus which corresponds roughly to the internalpressure in the reaction chamber.

The cartridge is made preferably of polypropylene (PP). This is an inertplastic material which requires no additional passivation of surfaces inorder in order to make possible temperature-controlled biologicalreactions (in particular the PCR process) in the reaction chamber. Thecartridge may however also be made of another suitable plastic such ase.g. cycloolefincopolymer (COC) or another suitable material.

The transparent plastic film may be provided on its side facing thebiochip with an adhesive or bonding layer which can be activated when itcomes into contact with the sample solution. When the plastic film ispressed on to the biochip it adheres to the biochip, thereby preventingsample solution from entering between the biochip and plastic film. Thisadhesive or bonding layer is preferably provided on that area of thefilm which is not in contact with the area of the biochip containing itsspots. The adhesive or bonding layer is thus provided so as to runaround the active area of the biochip.

The invention will be explained with the aid of an embodimentillustrated in the drawings. The drawings show in schematic form in:

FIG. 1 a perspective view of a casing of a cartridge according to theinvention

FIG. 2 a view of the casing of FIG. 1 in a partly-sectioned side viewtogether with a stopper,

FIG. 3 a cartridge according to the invention, showing a sectional viewcorresponding to line A-A plotted on the casing of FIG. 2,

FIG. 4 a section through the cartridge and a scanning chamber,

FIG. 5 the filling process of the cartridge with the stopper not inplace,

FIG. 6 the filling process of the cartridge with the stopper pressed in,

FIG. 7 a circuit board of the cartridge according to the invention witha heating/measuring structure,

FIG. 8 a schematic section through the reaction chamber and the scanningchamber, with a film fitting up against a biochip,

FIG. 9 a sectional view of the scanning chamber of FIG. 4 and acompressed air supply source connected to it, and

FIG. 10 a section through the stopper and a filling nozzle of thecartridge.

A cartridge 1 with a biochip 30 will be described with the aid of FIGS.1-4.

The cartridge 1 has an elongated roughly bowl- or trough-like casing 2made e.g. of plastic by injection moulding. The casing 2 comprises abase wall 3 and an all-round side wall 4 with two long sides 5 and twoend faces 6. The side of the casing 2 on which the side wall 4 islocated is designated as the inside 7.

On the side of the base wall 3 facing away from the side walls 4, acircuit board 8 is fixed to the casing 2. This side of the casing 2 isdesignated as the outside 9.

At one end face 6 of the cartridge 1, a tubular section extending in theaxial direction is designated as the filling nozzle 10. The fillingnozzle 10 is formed integrally as one piece with the casing 2.

The filling nozzle 10 has a filling section 11 and a compression section12, with the filling section 11 located adjacent to the free edge of thefilling nozzle 10 and the compression section 12 situated between thefilling section 11 and the rest of the body of the cartridge 1 (FIG. 2).

The filling section 11 has a greater clear width than the compressionsection 12. Provided as a transition between the filling section 11 andthe compression section 12 is a chamfer 13. This chamfered section 12has, roughly in the middle, a sealing point 14, from which point astopper 15 tightly seals the filling nozzle 10 (FIG. 3).

The compression section 12 is a cylindrical section 16 with circularcross-section, which merges into a funnel-shaped section 17, at the endof which is a filling hole 18. The filling hole 18 is connected to areaction chamber 22 via a filling line section 20.

A filling indicator may be provided in the cylindrical section 16 of thecompression section 12. The filling indicator 19 may be in the form of amarking or calibration mark applied to the inside or outside of thefilling nozzle 10. It is also possible to provide a viewing panel orother suitable means in the filling nozzle 10, for use in checking thesample quantity inserted in the filling nozzle 10.

From the filling hole 18, the filling line section 20 extends roughlyperpendicular to and through the base wall 3. At the outside 9 of thebase wall 3, the filling line section 20 is in the form of a half-openrecess, wherein the open side is bounded by the circuit board 8.

Provided roughly in the centre of the base wall 3 is a rhomboid-shapedthrough hole 21 which bounds the reaction chamber 22.

One tip of the rhomboid-shaped through hole 21 points in the directionof the filling nozzle 10 and an opposite tip of the rhomboid-shapedthrough hole 21 points away from the filling nozzle 10.

The filling line section extends around the rhomboid-shaped through hole21 and leads into the tip of the rhomboid-shaped through hole 21 whichfaces away from the filling nozzle 10.

Connected to the tip of the rhomboid through hole 21 oriented in thedirection of the filling nozzle 10 is a compensation line section 23.The compensation line section 23 is formed in the outside 9 of the basewall 3 as a half-open recess, which in turn is bounded by the circuitboard 8. The compensation line section 23 extends on the side of therhomboid through hole 21 opposite the filling line section 20 around thethrough hole 21 and leads into one tip of of a half-cone-shapedcompensation chamber 24.

The half-cone-shaped compensation chamber 24 is in the form of a recess,open on one side, on the underside 9 of the base wall 3. Ahalf-cone-shaped section of the base wall 3 arches over the circuitboard 8 and bounds the compensation chamber 24, which extends to aroundthe height of the side walls 4.

Provided in the compensation line section 23, adjacent to the tip of therhomboidal through hole 21, is a circular recess. The recess is closedon the inside 7 of the base wall 3 by a transparent hemisphere 26moulded integrally to the base wall 3. Adjacent to the hemisphere 26 isa polished cone 25, extending into the compensation line section 23. Thetip of the cone 25 points towards the circuit board 8 and has an angleof 90°. The plastic material of the casing 2 is transparent andpreferably translucent. The hemisphere 26 forms a lens and is preferablypolished on its surface. The hemisphere 26 and the cone 25 form afilling indicator 27 for the reaction chamber 22.

If the filling indicator 27 is not filled with sample solution then thehemisphere 26, owing to the total reflection of the incident light, hasa light colour. If the filling indicator 27 is filled with sample fluid,then the cone 25 is surrounded by sample fluid, so that the the totalreflection is attenuated. This causes the circuit board 8 locatedbeneath to become visible, and the filling indicator appears dark.

On the inside 7 of the base wall 3, the reaction chamber 22 and thethrough hole 21 respectively are bounded by a flexible transparent film28. The film 28 is affixed to the base wall 3. The 28 has a thickness ofaround 127 μm. The film 28 is made preferably from a fluorinatedethylene-propylene copolymer (e.g. FEP) or another suitable plastic. Theclearance from the circuit board 8 comes to around 0.9 mm, correspondingroughly to the thickness of the base wall 3. The flexible transparentfilm 28 serves as a window for the optical measurements of the samplesolution.

A continuous seal 29 is provided on the film 28 in the area around thethrough hole 21.

In the area of the rhomboidal through hole 21 and the reaction chamber22, the circuit board 8 forms a boundary wall of the same. In the areaof the filling line section 20, the compensation line section 23, thefilling indicator 27 and the compensation chamber 24, the circuit board8 seals tightly with these recesses in such a way that they are boundedat the bottom and form a continuous, communicating and self-containedpassage structure.

The circuit board 8 contains contact faces, a digital storage medium(e.g. an EEPROM) and an internal heating/measuring structure 32, whichwill be explained in detail below. Instead of the circuit board 8, it isalso possible to provide for heating and cooling a plate of a materialwith good thermal conductivity, such as e.g. aluminium or copper. Thisplate can then be tempered from the outside using a heating/coolingdevice.

In roughly the centre of the through recess, a biochip 30 is mounted onthe circuit board 8. The biochip has an active side, on which probes areimmobilised at predetermined spots. The active side of the biochip facestowards the film 28. The biochip 30 has a height of around 0.7 mm and anumber of spots=M×N. To avoid optical back reflections and undesiredfluorescent radiation from the circuit board 8, the biochip 30 isoptically opaque on the reverse and is not fluorescent, e.g. coated withblack chrome.

In producing the cartridge 1, firstly the biochip 30 is fixed to thecircuit board 8 and then the circuit board 8 is bonded to the casing 2.The bond between the circuit board 8 and the biochip 30 is made with anadhesive bonding layer, such as e.g. a suitable adhesive tape (suitablefor biological reactions) or else a silicone adhesive.

The circuit board 8 with affixed biochip 30 is then aligned with thecasing 2 and fixed to it. A durable, temperature- and water resistantbond may be realised e.g. by using biologically-compatible adhesivetape, silicone adhesive, by laser welding, by ultrasonic welding or bybiologically-compatible adhesives.

At the same time it is possible to cover a large area of the circuitboard 8 with the adhesive tape (or adhesive), to affix the biochip 30over the heating/measuring structure, and then to align the casing 2with the biochip 30 and to fix the circuit board 8 over the wholesurface of the casing 2.

A second option for bonding the circuit board 8, biochip 30 and casing 2lies in the targeted flat bonding of the biochip 30 to the circuit board8 (adhesive only beneath the biochip 30) and the subsequent fixing ofthe casing 2, wherein the adhesive layer is provided only outside thereaction chamber 22. With this type of bonding, the transfer of heatfrom the heating/measuring structure in the circuit board 8 into thereaction chamber 22 is more efficient.

The reaction chamber 22 is bounded between the circuit board 8 and thefilm 28 and by the edge of the rhomboidal through hole 21. The circuitboard 8 is provided with the heating/measuring structure in order toheat up the biochip 30 to a predetermined temperature and to measure thetemperature. The film 28 serves as a viewing panel for an opticaldetection device, which is able through the film 28 to make an opticalscan of spots on the biochip 30. Since the 28 is flexible, it may beplaced on the surface of the biochip 30 in order to scan it.

To close the filling nozzle 10 and to create the overpressure in thereaction chamber 22, a stopper 15 is provided. The stopper is madepreferably of a thermoplastic elastomer (TPE). The stopper 15 iscylindrical with a circular cross-section. The outside diameter of thestopper 15 is such that it may be held virtually without play in thecompression section 12 of the filling nozzle 10. The cylindricalcompression section 12 of the filling nozzle 10 and the stopper 15 forma kind of cylinder-piston system. The stopper 15 is provided with a sealring 31 of the same material as and integrally moulded with the stopper.The stopper 15 may also be provided with a continuous groove, in which aseal ring 31 is located. The stopper 15 provides a fluid-tight seal ofthe hollow spaces of the cartridge 1 after approx. one-third of itslength has been inserted into the filling nozzle 10.

On the inside 7 of the casing 2 there are around the reaction chamber 22three means for guidance, centering and as a stop for a detection device49.

In the area of the tip of the rhomboidal reaction chamber 22 orientedtowards the filling nozzle 10, two opposite sections of the long sides 5of the side wall 4 are elevated relative to the remainder of the sidewall 4. These two sections are designated as stops 45. They are designedfor guidance and as stops for a scanning chamber.

In the area of the tip of the reaction chamber 22 oriented towards thecompensation chamber 24, a circular centering pin 44 is moulded on atright-angles. The centering pin 44 serves in conjunction with the stops45 for centering when the scanning chamber 40 is put in place and as astop for the scanning chamber 40.

The filling and compensation line sections 20, 23 are as short aspossible and are designed with the smallest possible cross-section, sothat the total volume is kept small and the necessary surplus of samplefluid is as low as possible. The line sections 20, 23 are however guidedaround the reaction chamber 22, so that in filling, in which the fillingnozzle 10 is arranged at the top, the reaction chamber 22 fills from thebottom. This ensures that the reaction chamber 22 is filled withoutcreation of bubbles. In operation, the filling nozzle 10 is at thebottom and the compensation chamber 24 at the top, so that sample fluidmay be displaced reversibly into the compensation chamber 24, while theair bubble during displacement is always above the sample fluid in thecompensation chamber 24.

The process of a temperature-controlled biological detection reactionrequires the setting of precise temperatures of the sample fluid in thereaction chamber. Here, in the course of a PCR, temperatures of e.g.between 30° C. and 98° C. are selected. The temperature distribution ofthe sample fluid should be homogeneous in the reaction chamber andtemperature changes (heating, cooling) should be made quickly.

Provided on the circuit board 8 (FIG. 7) is a heating/measuringstructure 32, which with current passed through the ohmic resistor actsas a heater. With this, the sample fluid in the reaction chamber 22 isheated to the required temperature T. The heating/measuring structure 32may at the same time be used as a temperature detector, by using theresistance characteristic R(T) to determine temperature.

A preferred embodiment of the layout of the circuit board 8 is shown inFIG. 7. The meandering heating/measuring structure 32 is formed by athin conductor path with a width of 60 μm and a thickness of 16 μm. Itis around 480 mm long. At room temperature it has an electricalresistance of around 6 to 8 ohms. The conductor path is made of copper,preferably copper with a purity of 99.99%. Copper of this level ofpurity has a thermal coefficient which, in the temperature rangerelevant here, is virtually constant. In its totality theheating/measuring structure 32 forms a rhombus with an edge length ofapproximately 9 mm. Conductor paths already exist which have a copperlayer with a thickness of 5 μm and on which structures with a width of30 μm are formed. A resistance of approximately 100 ohms to 120 ohms hasbeen obtained with conductor paths of this kind.

The biochip 30 has an edge length of only 3 mm, meaning that the rhombusformed by the heating/measuring structure 32 and a temperaturehomogenisation layer covers a greater area than the biochip 30.

The circuit board 8 may be provided with a temperature homogenisationlayer, which effects homogenisation of the temperature distribution onthe upper side of the circuit board 8. The temperature homogenisationlayer is a copper layer which is nickel-plated and provided with anadditional gold layer. The gold layer has the advantage that it is inertfor biological materials and therefore biological materials may comeinto direct contact with this layer in the reaction chamber. Thisreaction chamber 22 may therefore also be used for other experimentsbesides those involving the biochip. This homogenisation layer has goodthermal conductivity. Instead of a combined copper-nickel-gold layer, arelatively thick copper layer could also be provided.

A heating conductor path integrated in the circuit board 8 has a lowinherent thermal capacity. This makes it possible to achieve high ratesof heating of the sample fluid in the reaction chamber 22.

The end points of the meandering heating/measuring structure 32 merge ineach case into a very wide conductor path 33 and 34, which serve tosupply the heating current and themselves have only a low resistance dueto their considerable width. In addition, there is in each case afurther conductor path 35 and 36 connected to these two conductor paths33 and 34 in the area of the connection point of the meanderingheating/measuring structure.

These two additional conductor paths 35 and 36 are used to pick up thevoltage drop at the heating/measuring structure 32. The electricalcontrol of the heating/measuring structure 32 is described in detail inWO 2008/064865 A2. Reference is made to this document in respect of thecontrol of the heating/measuring structure 32.

The circuit board 8 has conductor paths 37 and corresponding contactpoints 38, 39 for the connection of an electrical semiconductor memory.This semiconductor memory is used to store calibration data for theheating device and also the data from the biological experiments to bemade with the biochip of the cartridge. This data is therefore storedwith no risk of confusion.

The dimensioning of the filling nozzle 10, the filling line section 20,the reaction chamber 22, the compensation line section 23, thecompensation chamber 24 and the stopper 15 of the cartridge 1 accordingto the invention will be described below with the aid of a typicalembodiment (FIG. 5, FIG. 6, FIG. 10).

The volume of the compensation chamber V_(A) under the assumption thatair behaves like an ideal gas, that the pressure build-up is isothermal,and the fluid sample is not compressible, is given by

${V_{A} = {{\frac{p_{0}}{p_{1} - p_{0}}\left( {V_{KB} + V_{R}} \right)} - V_{KA}}},$

wherein V_(KB) is the volume of the filling line section 20 (6.5 μl),V_(KA) the volume of the compensation line section 23 ( . . . μl), V_(R)the volume of the reaction chamber 22 (35.4 μl+8 μl), p₀ theenvironmental pressure (1 bar) and p₁ the increased pressure (1.4 bar).

To begin with, In calculating the height of the stopper 15 penetratinginto the compression section 12 a first penetration volume of thestopper V_(V) is calculated, which should penetrate into the compressionsection 12 of the filling nozzle 10 without the filling nozzle 10 beingsealed. This is therefore the volume which penetrates into thecompression section 12, until the stopper 15 seals with the compressionsection 12. The first penetration volume of the stopper V_(V) followsfrom the overall volume V_(t) (150.2 μl) of the filling nozzle betweenthe chamfer and the filling hole, less a surplus volume V_(P1) (10 μl)of the sample fluid which should remain in the filling nozzle, so thatwith movements of fluid no air can gain access to the filling linesection 20, a sample volume V_(P2) (49.9 μl), which is comprised of the35.4 μl referred to above, 6.5 μl for the filling line section 20 and 8μl for compensation for the bulging of the film 28, and an air reserveV_(L) of 30 μl in the 10 for any excess pipetted sample:

V_(V)=V_(t)−V_(P1)−V_(P2)−V_(L)

The air reserve V_(L) remains trapped between the sample solution andthe stopper 15. The first penetration depth h_(V) of the stopper 15 inthe compression section 12 therefore follows from the penetration volumeV_(V) divided by the cross-sectional area of the stopper A_(V):

$h_{V} = \frac{V_{V}}{A_{V}}$

A second penetration depth h_(s) specifies how far the stopper 15 mustpenetrate into the compression section 12 after sealing, so that theincrease of pressure to 1.4 bar is effected. Since the sample volumeV_(P2) (49.9 μl) is to be completely displaced from the filling nozzle10, all that remains in the filling nozzle 10 after the stopper has beenpressed in fully are the surplus volumeV_(P1) (10 μl) of the samplefluid and the air reserve V_(L), which is compressed on account of thepressure build-up. The volume V_(t′) displaced by the stopper 15 afterit has been fully pressed in thus follows from the following formula:

$V_{t^{\prime}} = {V_{t} - V_{P\; 1} - {\frac{p_{0}}{p_{1}}V_{L}}}$

The second penetration depth h_(s) of the stopper 15 in the compressionsection 12 therefore follows from the second penetration volume V_(t′)divided by the cross-sectional area of the stopper 15 A_(V):

$h_{S} = \frac{V_{t^{\prime}}}{A_{V}}$

The connection of the cartridge 1 to an apparatus for the analysis ofbiological samples using temperature-controlled biological reactionswill be described below.

This apparatus according to the invention comprises a scanning chamber40, a detection device 49 and a compressed air supply source 50.

The scanning chamber 40 is in the form of a tube. The tube 40 is placedwith an open sample side 41 on to the film 28 bounding the reactionchamber 22 or on to the seal 29 provided on top thereof.

The diameter of the tube 40 on the sample side 41 corresponds to thediameter of the seal 29 located on the film 28. The sample side 41 isbounded at the end by a sharp-edged continuous blade 42. The blade 42 isso designed that, when the scanning chamber 40 is placed on thecartridge 1, it penetrates into the seal 29 of the cartridge 1 andprovides a pressure-tight connection.

Provided on the sample side 41 of the tube 40 are recesses 43 toaccommodate the centering pin 44 and the two stops 45 of the cartridge1. By means of the stops 45 and the centering pin 44, the tube 40 ispositioned exactly on the cartridge 1 and its seal 29 respectively andover the reaction chamber 22. The stops 45 set the exact contactpressure with which the scanning chamber 40 rests pressure-tight on topof the cartridge 1.

Located at the end of the scanning chamber 40 opposite the sample side41 is part of an optical system 46. In the present embodiment e.g. twolenses 47 are provided.

Connected at around the middle of the tube 40 is a compressed air line48, in turn connected to a compressed air supply source 50. Thecompressed air supply source 50 is so designed as a cylinder/piston unitwith an electrically controlled linear drive, that the pressure at thepiston may be set precisely through the linear drive. Over thecompressed air supply source 50 and the compressed air line 48, thescanning chamber 40 may be pressure-loaded by compressed air in the areabetween the lenses 47 and the cartridge 1. By this means the film 28 isapplied flat to the biochip 30.

Located behind the two lenses 47 is a detection device 49 for scanningthe biochip 30.

In the present embodiment, three apertures 51 are provided in thescanning chamber 40.

The detection device 49 for readout of the biochip 30 is described indetail in WO 2007/135 091 A2, to which reference is hereby made. Thedetection device 49 comprises an LED with cast-on plastic optics, LEDoptics, illumination optics, an exciting filter, a dichroic mirror, anNA aperture, an emission filter, readout optics and a camera.

Preferably used as light source is a single high-performance LED withhigh optical power density and LED optics with high numerical aperture(NA). High-performance light-emitting diodes are light-emitting diodeswith a luminous flux of at least 35 lm. Preferably light-emitting diodeswith a luminous flux of at least 40 lm, at least 50 lm or at least 100lm are used. In the present embodiment the light-emitting diode LuxeonStar (red, 3 W electrical power consumption, 90 lm luminous flux) isused.

The light-emitting diodes used emit roughly punctuatedly light. By thiswe mean light-emitting diodes with an emitting surface not greater than1 mm×1 mm. Preferably the emitting surface does not exceed 0.5 mm×0.5mm. The smaller the emitting surface, the better the light may beconcentrated, with the individual light rays of the light rayconcentration aligned parallel to one another.

One advantage of an LED for the illumination of biochips also lies inthe variability of the wavelength. LEDs are available in the wholevisible spectrum with high efficiency. A further benefit of LEDs e.g.compared with lasers, for flat illumination, is that no intensitymodulations due to coherence occur at the surface.

For the typical dyes to be detected in the spots on the biochip, aspectrally well adjusted LED may be used as light source.

For Example:

-   -   dye Cy5 absorption maximum at 649 nm        -   excitation by LED LUXEON LD3 (colour red, max. at 630 nm)    -   dye Cy3 absorption maximum at 514nm        -   excitation by LED LUXEON LM3 (colour green, max. at 530 nm)    -   dye Alexa Fluor 532 absorption maximum at 532        -   excitation by LED LUXEON LM3 (colour green, max. at 530 nm)

The choice of an LED is made for a wavelength range LA_(E) which isrequired for fluorescence measurements (e.g. for dyes Cy5, Cy3, Alexa, .. . ). LEDs are small and compact with high efficiency and a long lifeexpectancy.

The camera has a flat CCD element as detector, so that a rectangular, inparticular square image may be recorded. The camera is so designed thatexposure times of more than 20 seconds are possible in its use. Normallythe maximum exposure time with such cameras is limited to a few seconds.As a rule the camera is exposed for 5 to 10 seconds to record the imageof the biochip. The longer the exposure time, the weaker thefluorescence signals which can be detected. The strength of thefluorescence signals depends heavily on the number of PCR cyclesperformed on the biochip. An individual PCR cycle lasts for around halfto several minutes. By means of long exposure times, which may beextended by quite a few seconds, a processing time of several minutescan be saved in the extent of the PCR cycles, and the overall processingtime may be kept short. These relatively long exposure times alsorepresent an advantage compared with conventional scanners, which scanthe individual spots in sequence, since with this procedure the exposuretime is not freely variable.

Through the dichroic mirror, the illumination optics are separated fromthe readout optics. The illumination optics are provided with a highnumerical aperture. The readout aperture is located in the beam path ofthe readout optics. This aperture reduces the numerical aperture of thereadout optics, by means of which a contrast-rich image with minimalimaging errors of the spots of the biochip is obtained on the camera.The numerical aperture of the readout channel should not exceed 0.25 andshould preferably be no greater than 0.15.

The two lenses 47 in the tube 40 are parts of both the illuminationchannel and also the readout channel.

The use of the apparatus for analysing biological samples usingtemperature-controlled biological reactions is described below (FIG. 8,FIG. 9).

The scanning chamber 40 is placed with its sample side 41 on the film ofthe cartridge 1. In the course of this, the recesses 43 of the scanningchamber 40 engage in the centering pin 44 and the two stops 45, by whichmeans the scanning chamber 40 is positioned and the contact pressure onthe seal 29 is limited. At the same time the blade 42 penetrates intothe seal 29 and ensures a pressure-tight connection.

The scanning chamber 40 is filled with air through the compressed airline. In this way an overpressure of between 2 and 4 bar is produced inthe space sealed between the optics and the transparent film 28. Due tothe overpressure, the transparent film 28 fits up against the biochip 30and displaces the fluorescence excess of the sample fluid over thebiochip 30.

The fluorescence in the rest of the sample chamber is screened off. Itis therefore possible to make a fluorescence image of the biochip 30.

A mixing (forced convection) of the sample has a positive effect on thespeed of reaction of the hybridisation/incubation, therefore thehybridisation/incubation time reduces and higher signal intensities arepossible. Such forced convection may be obtained by varying theoverpressure at the outside of the film.

Due to the internal overpressure of the cartridge 1 amounting to 0.4bar, the “window” film 28 is mechanically stressed, in particular duringheating-up, and would be subject to plastic deformation. This stress maybe neutralised by an external overpressure, also amounting to 0.4 bar.This is of advantage e.g. during amplification, in which the film issubject to considerable thermal stress.

In the case of the apparatus according to the invention it isadvantageous that extensive surface irregularities on the biochip 30,other than with rigid cover glass slides known from the prior art, haveno effect on the fluorescence signal.

On account of its lower thickness and the closeness of the film 28 tothe biochip 30, the optical quality of the film 28 has only slighteffect on the ability to evaluate the fluorescence signals.

The application of the film 28 to the biochip 30 is reversible, so thatreal-time PCR is possible.

In the cartridge or in the apparatus, a cooling device for cooling thereaction chamber 22 may also be provided.

In principle it is possible within the scope of the present inventionfor the filling nozzle and stopper also to be used in conjunction with acartridge, in which a flexible electrical circuit board is provided,wherein the viewing window may be rigid, e.g. in the form of a glasspanel.

LIST OF REFERENCE NUMBERS

1 cartridge 2 casing 3 base wall 4 side wall 5 long side 6 end face 7inside 8 circuit board 9 outside 10 filling nozzle 11 filling section 12compression section 13 chamfer 14 sealing point 15 stopper 16cylindrical section 17 funnel-shaped section 18 filling hole 19 fillingindicator 20 filling line section 21 through hole 22 reaction chamber 23compensation line section 24 compensation chamber 25 cone 26 hemisphere27 filling indicator 28 film 29 seal 30 biochip 31 seal ring 32heating/measuring structure 33 conductor path (heating current) 34conductor path (heating current) 35 conductor path (measuring current)36 conductor path (measuring current) 37 conductor path 38 contact point39 contact point 40 scanning chamber 41 sample side 42 blade 43 recess44 centring pin 45 stop 46 optics 47 lens 48 compressed air line 49detection device 50 compressed air supply source 51 aperture

1-15. (canceled)
 16. A method of filling a cartridge, wherein thecartridge comprises a reaction chamber and a biochip mounted in thereaction chamber, a filling nozzle connected so as to communicate withthe reaction chamber, and a compensation chamber connected so as tocommunicate with the reaction chamber, a compensation line section and afilling line section communicating among the reaction chamber, a fillinglevel indicator and the compensation chamber such that before filling,normal pressure exists within the compensation line section, the fillingline section, the reaction chamber, the filling nozzle and thecompensation chamber of the cartridge, wherein the reaction chamber, thecompensation chamber and the said line sections are sealed, wherein thefilling nozzle forms a free passage to the reaction chamber from outsidethe cartridge, and a stopper is provided which fits positively andtightly in the filling nozzle such that, when pressed in over a certaindistance, fluid is displaced from the filling nozzle towards thereaction chamber wherein the filling nozzle has a filling section, whichallows the biological sample to be introduced from outside, and whichleads into a cylindrical compression section, wherein the fillingsection has a larger cross-section than the compression section and thecompression section is connected to the reaction chamber via the freepassage, and the stopper is designed so that it is held tightly in thecompression section, wherein the reaction chamber is bounded on an upperside by a transparent, flexible film, which forms a viewing window,wherein the method comprises placing the filling nozzle facing upwards,filling a liquid biological sample into the filling nozzle, pressing astopper into the filling nozzle in order to force sample liquid into thereaction chamber, so that the reaction chamber is filled from bottom totop and an air bubble remains in the compensation chamber downstream ofthe reaction chamber.
 17. A method of filling a cartridge, wherein thecartridge comprises a reaction chamber and a biochip mounted in thereaction chamber, a filling nozzle connected so as to communicate withthe reaction chamber, and a compensation chamber connected so as tocommunicate with the reaction chamber, a compensation line section and afilling line section communicating among the reaction chamber, a fillinglevel indicator and the compensation chamber such that before filling,normal pressure exists within the compensation line section, the fillingline section, the reaction chamber, the filling nozzle and thecompensation chamber of the cartridge, wherein the reaction chamber, thecompensation chamber and the said line sections are sealed, wherein thefilling nozzle forms a free passage to the reaction chamber from outsidethe cartridge, and a stopper is provided which fits positively andtightly in the filling nozzle such that, when pressed in over a certaindistance, fluid is displaced from the filling nozzle towards thereaction chamber wherein the filling nozzle has a filling section, whichallows the biological sample to be introduced from outside, and whichleads into a cylindrical compression section, wherein the fillingsection has a larger cross-section than the compression section and thecompression section is connected to the reaction chamber via the freepassage, and the stopper is designed so that it is held tightly in thecompression section, wherein the reaction chamber is bounded on an upperside by a transparent, flexible film, which forms a viewing window,wherein the method comprises placing the filling nozzle facing upwards,filling a liquid biological sample into the filling nozzle, pressing astopper into the filling nozzle in order to force sample liquid into thereaction chamber, so that the reaction chamber is filled from bottom totop and an air bubble remains in the compensation chamber downstream ofthe reaction chamber.
 18. A method of analyzing a biological sampleincluding introducing a biological sample to an apparatus for theanalysis of biological samples using temperature-controlled biologicalreactions, the apparatus comprising: a cartridge for analyzingbiological samples, the cartridge comprising: a reaction chamber and abiochip mounted in the reaction chamber, a filling nozzle connected soas to communicate with the reaction chamber, and a compensation chamberconnected so as to communicate with the reaction chamber, a compensationline section and a filling line section communicating among the reactionchamber, a filling level indicator and the compensation chamber suchthat before filling, normal pressure exists within the compensation linesection, the filling line section, the reaction chamber, the fillingnozzle and the compensation chamber of the cartridge, wherein thereaction chamber, the compensation chamber and the said line sectionsare sealed, wherein the filling nozzle forms a free passage to thereaction chamber from outside the cartridge, and a stopper is providedwhich fits positively and tightly in the filling nozzle such that, whenpressed in over a certain distance, fluid is displaced from the fillingnozzle towards the reaction chamber wherein the filling nozzle has afilling section, which allows the biological sample to be introducedfrom outside, and which leads into a cylindrical compression section,wherein the filling section has a larger cross-section than thecompression section and the compression section is connected to thereaction chamber via the free passage, and the stopper is designed sothat it is held tightly in the compression section, wherein the reactionchamber is bounded on an upper side by a transparent, flexible film,which forms a viewing window. wherein the biochip has an active side andthe area of the cartridge opposite the active side of the biochip is inthe form of a transparent film and is arranged with clearance from thebiochip, a detection device, and a scanning chamber which may beconnected to the cartridge in a pressure-tight fashion, in the area ofthe film, wherein the detection device is connected to the chamber suchthat the biochip may be scanned through the film and a compressed airline leads into the scanning chamber, so that the film may be sopressurized by air pressure that it is pressed flat against the biochip,the method comprising: arranging the cartridge in the apparatus with thefilling nozzle facing downwards, conducting an optical scan of thereaction chamber, thereby creating an overpressure of 1 bar to 4 bar inthe scanning chamber such that the film fits up against the biochip andthe sample fluid is displaced from the reaction chamber towards thecompensation chamber, wherein an air bubble remains above the samplefluid in the compensation chamber.
 19. The method of claim 18, whereinno optical scanning takes place and the reaction chamber is heated, apressure is generated in the scanning chamber which corresponds roughlyto the internal pressure in the reaction chamber.
 20. A method ofanalyzing a biological sample using an apparatus for the analysis ofbiological samples using temperature-controlled biological reactions,the apparatus comprising a cartridge for analyzing biological samples,wherein the cartridge comprises: a reaction chamber and a biochipmounted in the reaction chamber, a filling nozzle connected so as tocommunicate with the reaction chamber, and a compensation chamberconnected so as to communicate with the reaction chamber, a compensationline section and a filling line section communicating among the reactionchamber, a filling level indicator and the compensation chamber suchthat before filling, normal pressure exists within the compensation linesection, the filling line section, the reaction chamber, the fillingnozzle and the compensation chamber of the cartridge, wherein thereaction chamber, the compensation chamber and the line sections aresealed, wherein the filling nozzle forms a free passage to the reactionchamber from outside the cartridge, and a stopper is provided which fitspositively and tightly in the filling nozzle such that, when pressed inover a certain distance, fluid is displaced from the filling nozzletowards the reaction chamber, wherein the filling nozzle has a fillingsection, which allows the biological sample to be introduced fromoutside, and which leads into a cylindrical compression section, whereinthe filling section has a larger cross-section than the compressionsection and the compression section is connected to the reaction chambervia the free passage, and the stopper is designed so that it is heldtightly in the compression section, wherein the reaction chamber isbounded on an upper side by a transparent, flexible film, which forms aviewing window, wherein the biochip has an active side and the area ofthe cartridge opposite the active side of the biochip is in the form ofa transparent film and is arranged with clearance from the biochip, adetection device, and a scanning chamber which may be connected to thecartridge in a pressure-tight fashion, in the area of the film, apiston/cylinder pump is provided as compressed air supply source, inorder to supply the scanning chamber with pressure, wherein thedetection device is connected to the chamber such that the biochip maybe scanned through the film and a compressed air line leads into thescanning chamber, so that the film may be so pressurized by air pressurethat it is pressed flat against the biochip, wherein the cartridge isarranged in the apparatus with the filling nozzle facing downwards,wherein an optical scan of the reaction chamber is conducted such thatan overpressure of 1 bar to 4 bar is created in the scanning chambersuch that the film fits up against the biochip and the sample fluid isdisplaced from the reaction chamber towards the compensation chamber,wherein an air bubble remains above the sample fluid in the compensationchamber.
 21. A method of analyzing a biological sample using anapparatus for the analysis of biological samples usingtemperature-controlled biological reactions, the apparatus comprising acartridge for analyzing biological samples, wherein the cartridgecomprises: a reaction chamber and a biochip mounted in the reactionchamber, a filling nozzle connected so as to communicate with thereaction chamber, and a compensation chamber connected so as tocommunicate with the reaction chamber, a compensation line section and afilling line section communicating among the reaction chamber, a fillinglevel indicator and the compensation chamber such that before filling,normal pressure exists within the compensation line section, the fillingline section, the reaction chamber, the filling nozzle and thecompensation chamber of the cartridge, wherein the reaction chamber, thecompensation chamber and the line sections are sealed, wherein thefilling nozzle folios a free passage to the reaction chamber fromoutside the cartridge, and a stopper is provided which fits positivelyand tightly in the filling nozzle such that, when pressed in over acertain distance, fluid is displaced from the filling nozzle towards thereaction chamber, wherein the filling nozzle has a filling section,which allows the biological sample to be introduced from outside, andwhich leads into a cylindrical compression section, wherein the fillingsection has a larger cross-section than the compression section and thecompression section is connected to the reaction chamber via the freepassage, and the stopper is designed so that it is held tightly in thecompression section, wherein the reaction chamber is bounded on an upperside by a transparent, flexible film, which forms a viewing window,wherein the biochip has an active side and the area of the cartridgeopposite the active side of the biochip is in the form of a transparentfilm and is arranged with clearance from the biochip, a detectiondevice, a piston/cylinder pump as a compressed air supply source, inorder to supply the scanning chamber with pressure, and a scanningchamber which is connected to the cartridge in a pressure-tight fashion,in the area of the film, wherein the detection device is connected tothe chamber such that the biochip is capable of being scanned throughthe film and a compressed air line leads into the scanning chamber, sothat the film is pressurized by air pressure that it is pressed flatagainst the biochip, wherein during the steps in which no opticalscanning takes place and the reaction chamber is heated, a pressure isgenerated in the scanning chamber which corresponds roughly to theinternal pressure in the reaction chamber.